作者感谢国立卫生研究院过去的资助和感谢迈克尔 r. 格林合成寡核苷酸。

注:图 1提供了硫代核苷酸突变的概述, 并总结了过程中的关键步骤。
1. 用非野生型核苷酸生成突变体掺杂 DNA 模板的库
<ol><li>合成 T7 底漆 (5 '-GTAATACGACTCACTATAG-3 ') 通过化学合成的 DNA 合成物。</li>
	<li>在与蛋白质结合部位相对应的 DNA 合成器上合成掺杂的寡核苷酸 (互补链)。在化学合成过程中使用适当的 phosphoramidites 混合物 (X 以下), 其比例为 90% a 作为野生型核苷酸和 10% T 作为非野生型核苷酸 (见有代表性的结果, 以更详细地说明这个比例)。 注: 在这里, 掺杂的寡核苷酸的序列是 5 '-GTTCACTACACTXGAXAXAXAXCAXCXAXAXGXTGCCCTATAGTGAGTCGTATTAC-3 '。带下划线的序列是 SXL 蛋白结合点的反向补充, 加上绑定站点外的额外核苷酸, 它提供了有用的控制, 确认并非 RNA 中的每一次变化都影响绑定, 以及加载控件以结合点内核苷酸的比较和规范化。在变压器前 mRNA14、15、16、17中存在 SXL 结合点序列 UUUUUGUUGUUUUUUUU, 用于设计所提出的方法。斜体序列是对 T7 引物序列的补充, 是体内转录的 T7 启动子。</li>
</ol>2. RNA 的合成
<ol><li>
在20µL 转录反应中合成 RNA, 如前所述18。
	<ol>
<li>混合 T7 转录缓冲器和1µM T7 寡核苷酸, 1 µM 掺杂寡核苷酸, 10 毫米 dithiothreitol (德勤), 2 毫米 GTP, 1 毫米每 ATP, CTP 和双绞线 (鸟嘌呤, 腺苷, 胞苷和苷三磷酸) 和 2 U/µL T7 RNA 聚合酶。</li>
		<li>另外, 在两个单独的离心管中, 0.167 毫米α硫氰酸盐或0.05 毫米α硫代双绞线将 phosphorothioates (见图 2中的示意图) 转化为 rna。 注: 对于替代协议, 添加0.2 毫米α硫氰酸 CTP 或0.2 毫米α硫 GTP, 以适当的转录反应含有掺杂寡核苷酸, 以测试两个剩余的核苷酸替代品。</li>
		<li>在37摄氏度孵化2小时的 RNA 合成反应混合物。</li>
	</ol>
</li>
	<li>添加2.5 µL 耐热碱性磷酸酶和2.5 µL 10xphosphatase 缓冲器的 RNA 样品。孵化这25µL 反应 10–30 37 摄氏度, 以去除 5 ' 磷酸盐。</li>
	<li>以80摄氏度为 2–5, 禁用碱性磷酸酶酶。</li>
	<li>Radiolabel 5 ' 末端去磷酸化 RNA (5 pmole) 使用1µL T4 核苷酸激酶和1µL γ32P ATP 在10µL 反应容量。在37摄氏度的30–60中孵化反应混合物。</li>
	<li>T4 核苷酸激酶酶通过加热65摄氏度, 以 20–30 min 灭活。</li>
	<li>凝胶通过电泳在10% 变性聚丙烯酰胺凝胶中纯化 RNA。显影在凝胶上定位 RNA。将含有核素 RNA 的凝胶切片, 用吸管尖挤压在离心管壁上, 浸泡在蛋白酶 K (PK) 缓冲 (100 毫米三, pH 7.5, 12.5 毫米 EDTA, 150 毫米氯化钠, 1% 钠月桂酸盐)。在室温下将管子从2小时旋转到一夜之间。</li>
	<li>离心胶浆, 丢弃凝胶, 收集缓冲液。</li>
	<li>用苯酚-氯仿等量量提取溶液两次, 一次用氯仿萃取, 收集水相。</li>
	<li>加入水相0.1 的3M 醋酸钠, pH 5.2, 载体 tRNA 或糖原, 2.5 的乙醇量。保持样品在-80 °c 为1小时。</li>
	<li>离心样品为5–10分钟在高速离心在 16873 x g。在不干扰 RNA 颗粒的情况下, 仔细去除缓冲液/乙醇溶液。</li>
	<li>用70% 乙醇和离心机清洗颗粒 2–5, 小心去除乙醇。在空气中干燥颗粒。</li>
	<li>并用重悬20–50µL 二乙基焦碳酸 (DEPC) 处理的水中的颗粒。存储在-20 °c, 直到使用。 注意: 使用适当的预防措施和有机玻璃防护罩, 用核素 RNA 执行所有步骤以防止放射性。目前, 自旋柱更常用, 更方便去除未合并的放射性, 作为一种替代的凝胶纯化 RNA。</li>
</ol>3. 蛋白质结合反应和束缚 RNA 的分离
<ol><li>表达重组蛋白, 并从大肠杆菌中纯化。</li>
	<li>用分光光度法或在已知蛋白质标准、牛血清白蛋白 (BSA) 旁边的 SDS-聚丙烯酰胺凝胶中分离来估计重组蛋白浓度。用考马斯亮蓝 R-250 染色凝胶, 可视化感兴趣的蛋白质和 BSA 标准。定量蛋白质与已知数量的 BSA 稀释在同一凝胶上进行比较。 注: 将重组蛋白贮存在-80 摄氏度, 直到使用和稀释20毫米 4-(2-羟乙基) 哌嗪-1-磺酸 (HEPES), pH 8.0, 1 毫米 dithiothreitol (德勤), 0.2 毫米乙二胺二乙酸 (EDTA), 0.05% NP-40, 20% 甘油。使用 0.5–1.0 mM 蛋白酶抑制剂 phenylmethane 磺酰氟 (PMSF) 是可选的。</li>
	<li>执行 RNA 结合反应 (20–100µL) 在10毫米三盐酸, pH 值 7.5, 1 毫米, 50 毫米氯化钾, 0.5 单位/µL RNase 抑制剂, 0.09 µg/µL 乙酰牛血清白蛋白, 1 毫米 EDTA, 0.15 µg/µL tRNA, 5 '-端核素 RNA, 6 µL 适当浓度 (a浓度在50% 的 RNA 结合蛋白质) 的蛋白质。 注意: 这个绑定缓冲区适用于三 RNA–binding 蛋白 (SXL, U2AF65, 和肺结核), 但必须标准化的蛋白质的兴趣。在经验上估计蛋白质浓度, 使用各种稀释为给定的蛋白质准备, 这是需要得到大约 50% RNA 捆绑。这种情况或Kd 表示 RNA 结合曲线最敏感的部分。</li>
	<li>在25摄氏度 (或冰上) 孵化20–30的蛋白质结合反应。</li>
	<li>
使用以下两种方法之一将蛋白质绑定的 RNA 分数与未绑定分数分开:
	<ol>
<li>硝化棉过滤器装订<ol>
<li>将结合反应 (20–100µL) 应用到在室温下连接到真空歧管的硝化纤维过滤器上。 注意: 只有 RNA 蛋白复合体保留在过滤器上, 而未绑定 rna 流经过滤器。与凝胶流动转移试验相比, 过滤结合方法允许更高的束缚 RNA 的恢复并且是快速和简单的。通过将 DEAE 膜置于硝化棉过滤器下面, 也可以收集未绑定的 rna 分数或游离 rna, 以与束缚分数进行比较。</li>
			<li>将含有保留放射性 RNA 的硝化棉过滤器的部分切成小块, 放入离心管中, 浸泡在足够的 PK 缓冲器中 (300–500µL 含10–20µg PK) 浸泡过滤片, 并从过滤器中洗脱 RNA。为 2–3 h 或过夜。</li>
			<li>用苯酚-氯仿、氯仿和收集水相萃取。添加醋酸钠和乙醇, 并在冷冻后, 离心, 洗涤, 干燥和并用重悬 RNA 在 DEPC 处理后的水中孵化。按照上文步骤2.10 至2.14 中详细说明的步骤操作。</li>
		</ol>
</li>
		<li>凝胶流动转移<ol>
<li>在开始粘结反应之前, 在 0.5x (标准的三硼酸-EDTA 缓冲器) 中制备和聚合5% 本地聚丙烯酰胺凝胶 (60:1 丙烯酰胺: 双丙烯酰胺), 作为过滤器结合方法的替代品。</li>
			<li>在寒冷的房间 (4 摄氏度), 在 250 V 预运行凝胶15分钟。</li>
			<li>将每个结合反应 (以上) 加载到上述预运行凝胶的分离井中。 注: 蛋白质贮存缓冲液为试样的入井提供了足够的甘油。</li>
			<li>根据 rna 大小和特异蛋白, 在冷室中用凝胶电泳将蛋白结合的 RNA 分离为250伏, 1 到2小时。</li>
			<li>显影在凝胶上定位 RNA 蛋白复合物的位置。对含有 RNA 蛋白复合物的凝胶切片进行消费。通过粉碎凝胶切片并浸泡在 PK 缓冲器中, 洗脱从凝胶切片中提取 RNA。</li>
			<li>用苯酚-氯仿、氯仿和收集水相萃取。加入醋酸钠和乙醇, 在冷冻、离心、洗涤、风干和并用重悬 RNA DEPC 处理后的水中孵化。按照上文步骤2.10 至2.14 中详细说明的步骤操作。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 用于检测突变核苷酸位置的碘裂硫代核苷酸产品分析
<ol><li>在20µL DEPC 处理的水中添加1毫米碘, 其中含有多达10µg 载体 tRNA 在硫代核苷酸公司的地点切割 rna (绑定和总 rna)。室温孵育5分钟。 注: 有关碘解裂的进一步细节, 略有不同的情况-7% (v/v) iodoethanol, 加热95摄氏度为3分钟-可以在吉什 & 埃克施泰因 198819中找到。</li>
	<li>如上文所述, 加入醋酸钠/乙醇沉淀裂解 RNA。并用重悬用于变性凝胶的负载染料中。在15–20% 变性聚丙烯酰胺凝胶中, 用电泳法将样品加热并载入分离 RNA 片段。</li>
	<li>将聚丙烯酰胺凝胶暴露在 x 射线胶片上。</li>
	<li>使用显影检测绑定分数与总池中的带区。 注: 使用排气罩中的碘执行所有步骤。对于这里显示的 RNA 分离, 采用楔形凝胶, 通过在凝胶底部插入一个额外的英寸长的间隔, 使两个垫片的厚度加倍, 从而实现。此形状允许较短的 RNA 片段之间更均匀或更接近的间距。另外, 可以使用 phosphorimager, 而不是 X 射线胶片来检测和定量放射性物质。</li>
</ol>

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撰文人没有宣布任何竞争的金融利益。

突变一直被用来表征蛋白质结合部位。首先, 一系列突变体可以构造和单独测试的结合检测, 以分析其对结合亲和性的影响。虽然标准突变方法提供了一种分析多个序列的方法, 但标准方法中涉及的多个步骤, 如构造突变体和对每个变种人执行一系列绑定反应, 都是费力和耗时的, 可能不会允许饱和诱变, 特别是对较长的序列。第二, 序列可以是随机的和池用于多周期的结合和聚合酶链反应 (PCR) 放大。这些绑定蛋白质的序列必须被克隆和排序, 以便从协商一致序列中识别和验证绑定站点。尽管如此, 这种迭代绑定和放大选项涉及多个步骤, 并且在确定绑定站点中重要的残滓方面很费力。此外, PCR 所固有的重复序列放大可能引入序列偏倚。第三, 从随机池中选择的序列可以使用更快的高吞吐量排序选项进行排序, 尽管成本相对较高。因此, 为了克服多种步骤、费力和/或昂贵方法的这些限制, 我们设计了一个更快、更廉价、最重要的单步骤方法, 在这里描述, 以完成绑定站点 (PTM) 的饱和诱变。
此方法的关键步骤包括识别或标记非野型核苷酸。我们使用的硫代核苷酸核苷酸, 其中一个氧在磷酸盐主干是取代硫, 如描述 (图 2)。其优点是, 碘可用于化学切割硫代核苷酸核苷酸的骨干。因此, 含有硫代核苷酸骨干的 RNA 基片的碘裂解产生一个测序式阶梯, 在这个位置上, 卵裂部位是四基地之一的代表或标记。对未绑定和总分数的比较确定了优先排除在绑定分数中的残留物, 因此对蛋白质绑定很重要。相比之下, 对绑定不重要的残滓在两个分数之间保持同样的分布。这种方法可用于定义任何 RNA 结合蛋白的结合部位。
总之, 这一步饱和诱变方法, 结合掺杂, phosphorothioates 和碘裂解, 提供了一个强大的手段, 以描述任何绑定网站的 RNA。但是, 这种方法要求绑定站点在突变之前已经知道。此外, protein–binding 条件需要对每种蛋白质进行优化。需要强调以下预防措施。必须执行 rna 处理的预防措施, 例如戴手套、在 DEPC 处理的水中制备溶液和缓冲液、热处理吸管尖和管子, 以及专门用于 rna 的设备, 以避免 RNAses。同样, 涉及放射性防护的护理、放射性物质的辐射监测是必不可少的, 在使用危险化学品时使用排气罩是必要的。

基因调控在生物学中起着重要的作用。基因可以调节在转录水平, 前 mRNA 剪接, 3 ' 末端形成, RNA 出口, 翻译, mRNA 定位, 衰变, 后转化/稳定性等. DNA 和 RNA 结合蛋白在基因中起关键作用调节。虽然分子遗传学分析已经确定了许多调控蛋白, 但它们中只有一小部分被完全用于细胞功能或体内的结合部位。系统进化序列分析和突变提供了互补的方法来表征 DNA 或 RNA-蛋白质相互作用。
RNA 结合蛋白在发育过程中很重要, 包括性分化。果蝇蛋白性致死 (SXL) 或主性开关蛋白不在男性, 但存在于女性。它能识别在体细胞1,2 中, 苷的序列或嘧啶-在下游前 mRNA 目标 (变压器,性致死和男性特定的 lethal2) 附近的特定剪接点. ,3,4。此外, 它通过结合苷丰富的 polyadenylation 增强器序列在基本(e (r)) 成绩单5,6中约束 polyadenylation 站点切换。SXL 可能对仍有待确定的女性生殖中的其他目标进行调节, 1、7、8、9、10、11、12,13。
通常, 绑定站点的特征包括突变, 例如, 通过删除或替代单个或多个核苷酸。每个突变结合点, 相对于野生类型的 RNA 序列, 然后分析使用一系列的蛋白质浓度, 以确定其结合亲和性 (Kd 或平衡离解常数) 的蛋白质的利益;Kd 是获得 50% RNA 结合所需的蛋白质浓度。这一劳动密集型的详细诱变过程涉及对众多突变体的产生和分析, 即三种非野生型核苷酸用于结合部位的每个位置。因此, 需要一种替代的方法, 以更快, 更简单, 廉价的饱和突变的蛋白质结合点在 RNA。
在这里, 我们描述了一步方法, 允许在 RNA 的蛋白质结合点的饱和诱变。它涉及掺杂 DNA 模板与非野生型核苷酸在结合部位, 合成单独 rna 与每个硫代核苷酸核苷酸, 并分离的束缚分数后孵化与蛋白质。非野生型核苷酸的干扰会导致它们在蛋白质束缚分数上的优先排斥。这是由凝胶电泳监测后, 选择性化学分裂与碘磷酸二酯债券含有 phosphorothioates (硫代核苷酸诱变或 PTM)。这种单步饱和诱变方法适用于 RNA 中任何蛋白质结合部位的表征。

<table><tbody><tr><td>Uridine 5&amp;rsquo; a-thio triphosphate</td><td>NEN (Boston, Massachusetts)</td><td>NLP-017</td><td></td></tr><tr><td>Adenosine 5&amp;rsquo; a-thio triphosphate</td><td>NEN (Boston, Massachusetts)</td><td>NLP-016</td><td></td></tr><tr><td>Vacuum manifold</td><td>Fisher Scientific</td><td>XX1002500</td><td>Millipore&amp;nbsp;25&amp;nbsp;mm Glass Microanalysis Vacuum Filter</td></tr><tr><td>Vacuum manifold</td><td>Millipore</td><td>XX2702552</td><td>1225 Sampling Vacuum Manifold</td></tr><tr><td>Nitrocellulose</td><td>Millipore</td><td>HAWP</td><td></td></tr><tr><td>Nitrocellulose</td><td>Schleicher &amp;amp; Schuell</td><td>PROTRAN</td><td></td></tr><tr><td>Dephosphorlyation Kit</td><td>NEB</td><td>M0508</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>Proteinase K</td><td>NEB</td><td>P8107S</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>NEB</td><td>M0251S</td><td></td></tr><tr><td>RNasin</td><td>Promega</td><td></td><td>RNase inhibitor</td></tr><tr><td>Glass Plates</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>Gel Electrophoresis equipment</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>X-ray films</td><td>Standard</td><td>Standard</td><td></td></tr><tr><td>Polyacrylamide gel solutions</td><td>Standard</td><td>Standard</td><td></td></tr></tbody></table>

a novel saturation mutagenesis approach: single step characterization of regulatory protein binding sites in rna using phosphorothioates

基因调控在发展中起着重要的作用。大量的 DNA 和 RNA 结合蛋白结合其靶序列的高特异性来控制基因表达。这些调控蛋白控制基因表达无论是在 DNA 水平 (转录) 或在 RNA 水平 (前 mRNA 剪接, polyadenylation, mrna 传输, 衰变和翻译)。规范序列的识别不仅有助于了解基因是如何开启或关闭的, 而且还能理解哪些下游基因是由特定的调控蛋白调控的。在这里, 我们描述了一步方法, 允许在 RNA 的蛋白质结合点的饱和诱变。它涉及掺杂 DNA 模板与非野生型核苷酸在结合部位, 合成单独 rna 与每个硫代核苷酸核苷酸, 并分离的束缚分数后孵化与蛋白质。非野生型核苷酸的干扰会导致它们在蛋白质束缚分数上的优先排斥。这是由凝胶电泳监测后, 选择性化学分裂与碘磷酸二酯债券含有 phosphorothioates (硫代核苷酸诱变或 PTM)。这种单步饱和诱变方法适用于 RNA 中任何蛋白质结合部位的表征。

利用掺杂的饱和诱变原理:
对于野生型和其他核苷酸的适当摩尔比, 如果只分析一个位置, 则使用所有四核苷酸的相等混合物。但是, 如果同时对多个位置进行分析, 则必须调整非野类型与野型核苷酸的比值,即减少。否则, 除了单一的替代, 这是需要的, 也将有多个非野生型核苷酸在一个分子的模板, 排除了对单核苷酸替代效应的分析。因此, 作为一个经验法则, 使用非野生类型的比例为 1/n 的野生型核苷酸, 其中 n 是要分析的位置数。在这里, 我们同时分析了10个位置, 并使用含有90% 的野核苷酸和10% 的非野生型核苷酸的混合物掺杂 DNA 模板。单独的转录反应完成, 其中每个转录反应是执行与四 phosphorothioates 之一。因此, 每个反应监测一个特定的核苷酸在掺杂的位置。
因干扰而划分的原则:
在这里提出的突变方法中, rna 在每个分子平均少于一个硫代核苷酸残渣。在结合过程中, RNA 分子在蛋白质束缚分数和未绑定分数之间划分。由于一个特定的核苷酸链接到一个硫代核苷酸的联系, 硫代核苷酸骨干联系的分裂是一个特定的核苷酸存在的读数在该位置。预计在任何给定的位置, 当核苷酸被改变, 它可能对绑定没有影响, 或者它可能会抑制部分或完全的绑定。如果特定核苷酸的存在对绑定没有影响, 它将在蛋白质绑定和未绑定分数之间平等地划分。然而, 如果一个特定的核苷酸在某一位置干扰蛋白质结合, 它将优先排除在蛋白质绑定分数。在凝胶电泳后, 同一反应中各部位的干扰程度均可定量监测。这个概念在示意图中被说明 (图 3)。在总 RNA 分数 (车道 T), 带强度近似地相等为所有掺杂的位置 (乐队 1, 3–7)。在蛋白质束缚 RNA 分数 (车道 B), 在位置 1, 4 和 7, 核苷酸没有作用对捆绑。但是, 在位置3和 6, 它干扰绑定, 因而被排除在绑定分数之外。在位置 5, 干涉是部份的。因此, 比较四配对车道, T 和 B 为每个核苷酸, 允许分析所有四核苷酸。应该指出的是, 某一位置的野生型核苷酸反映了 RNA 主干中硫在蛋白质结合上的作用。
伴随的 autoradiograph (图 4) 显示了两对 (α硫氰酸 A 和α-硫氰酸盐 U) 车道从变性凝胶 (T 为总水池和 B 为束缚的分数)。可以通过比较每对车道 (T 和 B) 之间每个位置的波段强度来进行一些观察。首先, 信号的大部分是在凝胶的顶部,即, uncleaved 产品, 为α硫氰酸 A 车道和α硫氰酸 U 车道。第二, 对于α硫的一对车道, 几个带 (碘裂解产品) 是相同的绑定和总 RNA 分数, 例如, 带1以上;在α硫氰酸 A 车道的结合部位内的相关带编号为参考。第三, 在凝胶底部的带比通常的顺序凝胶更紧密地间隔 (例如,带低于 6)。第四, 在总 rna 分数中存在几个带, 但在绑定 rna 分数中没有或显著减少 (例如,带1、2、3和 5)。第五, 对于α硫 U 车道对, 而大多数波段是可比的总和约束车道, 有些乐队是相对较少激烈的绑定分数 (例如,乐队7和 8)。
这些观察为这一新方法的成功发展提供了证据, 并得出以下结论: 首先, 对于α硫氰酸 A 对车道和α硫代 U 对车道, 因为多数 RNA 是 uncleaved 的, 它提供证据仅一小部分RNA 含有修饰或硫代核苷酸的残留物。这一点很重要, 因为它确定 RNA 分子不含超过一硫代核苷酸的残留物。第二, 在两条车道之间的结合点外的几个波段相同或相似的强度, 对于α硫 A 和α硫氰酸, 表明加载是可比的两个车道, 允许比较容易之间的车道。第三, 更紧密的间隔带是实现, α硫 A 和α硫氰酸盐, 由楔形的凝胶 (顶部稀释和底部厚), 从而允许分析更长的序列读取和提供更高的分辨率。通常, 较短的片段更广泛地间隔在一个均匀厚度的凝胶。第四, 在束缚分数中某些带的消失或强度减弱表明非野生型核苷酸优先被排除在束缚分数 (α硫 A) 之外。在束缚车道内不同波段的相对强度也提供了关于不同地点非野生类型残留物干扰程度的定量信息。换言之, 突变体或非野生型核苷酸在特定位置干扰蛋白质结合。最后, 在一个非桥式氧 (α硫硫代核苷酸) 中测试骨干硫替代品的效果, 表明三个位置显示了从骨干 sulfurs (如6、7和7以下) 的微小影响。我们的综合结果表明, 结合点内的几个位置显示优先消失或减少特定波段的强度在绑定分数。这是由于基础而不是骨干替代, 表明蛋白质相互作用的特定基地在结合部位 (α硫 A)。同时, α-硫代 C 的加入也显示出与α硫 A 相媲美的干涉模式。然而, 只有3–4α硫代 G 替换显示小但可察觉的干扰集中围绕, 例如, 位置编号2和 3 (没有显示数据)。该方法用于揭示拼接阻遏 SXL 和一般拼接因子 U2 snRNP 辅助因子 (U2AF65) 与相同的前 mRNA 拼接信号序列 (polypyrimidine-通道) 在不同方式下绑定的差异15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57816/57816fig1.jpg"> 图 1: 硫代核苷酸诱变 (PTM) 关键步骤流程图.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57816/57816fig2.jpg"> 图 2: 两个核苷酸之间的硫代核苷酸联系示意图, 可以用碘化学法进行裂解。硫磺取代了一个非桥接磷酸盐氧。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57816/57816fig3.jpg"> 图 3: 由于干扰而划分的原则.假设 RNA 包含有代表性的α硫核苷酸在六个位置, 包括一个蛋白质结合的站点。蛋白质结合后, RNA 在硫代核苷酸的部位被碘吸收。绑定站点内和周围1–7的位置被任意编号以供文本引用。位置2或缺失带代表没有硫代核苷酸合并或不掺杂核苷酸。车道 T 是总 RNA 和车道 B 是蛋白质束缚的 RNA。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57816/57816fig4.jpg"> 图 4: 硫代核苷酸突变 (PTM) 方法允许在变压器前 mRNA 中存在 polypyrimidine-tract/3 的剪接点的饱和诱变.车道 T 是总 RNA 和车道 B 是蛋白质束缚的 RNA。α硫氰酸酯是指在α硫 ATP 存在的情况下合成的 RNA, 并在序列中识别掺杂位置与α硫 adenosines。三强的乐队对应于 adenosines 在序列在那些位置。α硫 U 代表在α硫双绞线存在时合成的 RNA, 并在序列中识别所有的 uridines, 包括掺杂的位置。左侧的垂直线标记 SXL 绑定站点。在绑定站点内1–8的位置被编号为参考目的, 并且在结果部分中易于描述。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57816/57816fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates at JoVE.com

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

结合特定 RNA 序列的蛋白质在基因表达中扮演重要角色。对这些结合部位的详细描述对于我们理解基因调控至关重要。本文介绍了 RNA 蛋白质结合部位饱和诱变的单步法方法。这种方法与 RNA 中的所有蛋白结合部位有关。

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

a-novel-saturation-mutagenesis-approach-single-step-characterization

Nanyang Technological University

Research

JoVE Journal

Genetics

6.5K 次观看.  University of Colorado at Boulder. 结合特定 RNA 序列的蛋白质在基因表达中扮演重要角色。对这些结合部位的详细描述对于我们理解基因调控至关重要。本文介绍了 RNA 蛋白质结合部位饱和诱变的单步法方法。这种方法与 RNA 中的所有蛋白结合部位有关。

视频: A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

的快速方法新颖的RNA结合蛋白隔离

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

科研

遗传学

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

寡肽竞争测定磷酸化位点测定

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

生物化学

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

定量细菌中蛋白质-RNA结合的测定

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

生物学

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical approaches to solving RNA structure, such as RNA crystallography or cryo-EM, have failed to keep up with the rapidly evolving field and the need for high-throughput solutions. Mutational profiling with sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach to infer the RNA structure from a base&#39;s reactivity with DMS. DMS methylates the N1 nitrogen in adenosines and the N3 in cytosines at their Watson-Crick face when the base is unpaired. Reverse-transcribing the modified RNA with the thermostable group II intron reverse transcriptase (TGIRT-III) leads to the methylated bases being incorporated as mutations into the cDNA. When sequencing the resulting cDNA and mapping it back to a reference transcript, the relative mutation rates for each base are indicative of the base&#39;s &#34;status&#34; as paired or unpaired. Even though DMS reactivities have a high signal-to-noise ratio both in vitro and in cells, this method is sensitive to bias in the handling procedures. To reduce this bias, this paper provides a protocol for RNA treatment with DMS in cells and with in vitro transcribed RNA.

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods.

使用硫酸二甲酯探测RNA结构 体 外 和细胞测序突变分析

The role of RNA structure in virtually any biological process has become increasingly evident, especially in the past decade. However, classical ...

An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying the binding partners of an RNA of interest remains of high importance to unveil the mechanisms behind many cellular processes. However, RNA molecules might interact transiently and dynamically with some RNA-binding proteins (RBPs), especially with non-canonical ones. Hence, improved methods to isolate and identify such RBPs are greatly needed.
To identify the protein partners of a known RNA sequence efficiently and quantitatively, we developed a method based on the pull-down and characterization of all interacting proteins, starting from cellular total protein extract. We optimized the protein pull-down using biotinylated RNA pre-loaded on streptavidin-coated beads. As a proof of concept, we employed a short RNA sequence known to bind the neurodegeneration-associated protein TDP-43 and a negative control of a different nucleotide composition but the same length. After blocking the beads with yeast tRNA, we loaded the biotinylated RNA sequences on the streptavidin beads and incubated them with the total protein extract from HEK 293T cells. After incubation and several washing steps to remove nonspecific binders, we eluted the interacting proteins with a high-salt solution, compatible with the most commonly used protein quantification reagents and with sample preparation for mass spectrometry. We quantified the enrichment of TDP-43 in the pull-down performed with the known RNA binder compared to the negative control by mass spectrometry. We used the same technique to verify the selective interactions of other proteins computationally predicted to be unique binders of our RNA of interest or of the control. Finally, we validated the protocol by western blot via the detection of TDP-43 with an appropriate antibody. 
This protocol will allow the study of the protein partners of an RNA of interest in near-to-physiological conditions, helping uncover unique and unpredicted protein-RNA interactions.

Here, we present an optimized in vitro method to uncover, quantify, and validate protein interactors of specific RNA sequences, using total protein extract from human cells, streptavidin beads coated with biotinylated RNA, and mass spectrometry analysis.

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

Protein-RNA interactions regulate gene expression and cellular functions at transcriptional and post-transcriptional levels. For this reason, identifying ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

摘要

探索更多视频

此视频中的章节

的快速方法新颖的RNA结合蛋白隔离

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

寡肽竞争测定磷酸化位点测定

定量细菌中蛋白质-RNA结合的测定

探索序列空间,识别调节RNA结合蛋白的结合位点

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

使用硫酸二甲酯探测RNA结构体外和细胞测序突变分析

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

摘要

探索更多视频

此视频中的章节

的快速方法新颖的RNA结合蛋白隔离

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

寡肽竞争测定磷酸化位点测定

定量细菌中蛋白质-RNA结合的测定

探索序列空间,识别调节RNA结合蛋白的结合位点

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

使用硫酸二甲酯探测RNA结构 体 外 和细胞测序突变分析

使用短生物素化RNA对RNA结合蛋白进行优化的定量下拉分析

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

使用硫酸二甲酯探测RNA结构体外和细胞测序突变分析